Mrs. Brisby (Frisby in the original book) is a very shy, timid, and lonely mouse widow whose family is threatened by a premature warm spell, prompting the farmer to start his mowing early, endangering the cinder block she and her family call home. Normally she would simply pack up their things and move to a new spot in the field, but her younger son Timothy is too ill to be moved, forcing her to seek another alternative before it's too late. With the push of her local confidante, the Shrew, and the aid of a clumsy, love lorn crow named Jeremy, she goes to see the Great Owl, who, upon learning that she is the widow of a mouse named Jonathan Brisby, advises her to seek the aid of the mysterious rats who live in the rosebush outside the farmer's house. There, Mrs. Brisby learns of her deceased husband's past, and the secret of these rats, the Rats of NIMH, who agree to move her home before they abandon their underground colony for Thorn Valley, but Mrs. Brisby herself is endangered by the evil machinations of Jenner, a power hungry rat who seeks to overthrow the rats' leader Nicodemus so as to keep the rats in their underground colony, and will go so far as to endanger Mrs. Brisby's children to achieve his goals. Only Justin, the rats' gallant, Errol Flynn like captain of the guard, stands between Jenner and Mrs. Brisby, who must call upon the same courage her husband had if she is to save her family.
Mrs Brisby is a widowed mouse who lives on a farm owned by a man named Fitzgibbons. She and her 4 children, Martin, Theresa, Cynthia and Timmy live in a cinderblock in one of Fitzgibbons' fields. Every year, before Fitzgibbons plows the field to plant his crops, the family moves to their summer home to avoid the tractor. Nearby is another mouse, Dr Ages, who is the family doctor. Brisby visits Ages to get a remedy for Timmy, who has come down with pneumonia, potentially delaying the summer move.
Fitzgibbons decides one day to begin plowing the field early. Brisby desperately tries to disable the farmer's tractor and freezes while climbing up the mechanism. Auntie Shrew intervenes and pulls out the tractor's gas line, halting the machine. While Mrs Brisby sobs over her predicament, Auntie tells her to be braver for her family's sake and suggests she visit the Great Owl, the wisest creature in the forest. Brisby is hesitant, since owls eat mice, but she goes.
With the help of a new found friend, Jeremy, a crow hoping to meet Miss Right, she meets with the fearsome owl, who merely tells her that she must move her family. Brisby explains the complication with her son Timmy and the owl tells her he has no solution. As he leaves, he learns her last name and becomes more helpful because her husband, Jonathan, was well known throughout the woods. The Owl tells Mrs Brisby to go to the colony of rats that live in the farmer's rose bush and ask them for help. He specifically tells her to have the rats move her house to "the lee of the stone", the protected side of a large rock in the field that Fitzgibbons avoids when plowing.
Mrs Brisby sneaks into the rose bush and finds that the rats have built a very modern colony that makes use of electricity that they pilfer from Fitzgibbons. She also meets Dr Ages, who is astonished that she had met with the Great Owl and lived. They are discovered by the rats' captain of the guard, Justin, who takes them to the rats' Senate room. As they enter, a charismatic rat, Jenner, is speaking to the council about the plan of the rats' leader, Nicodemus, who wants to abandon the rose bush and move to a location called Thorn Valley, where they'll found a new colony based on their own labors, rather than stealing supplies from the farmer. Jenner is opposed to the idea and even suggest that they wage war against any humans who attempt to drive them out. Justin and Ages make Mrs Brisby's case to the council, who agree to the request because she is Jonathan Brisby's wife. In a sinister private meeting with his associate, Sullivan, Jenner plots to have Nicodemus killed during the operation and make it look accidental.
Justin takes Brisby to meet with Nicodemus. A very old and wise leader, he tells Mrs Brisby the rats' history: they were all lab animals imprisoned in NIMH, the National Institutes of Mental Health. Injected with experimental compounds, the rats developed intelligent brains and were able to escape their cages, along with several mice including Jonathan and Mr Ages. When they were trapped by a vent grating, Jonathan was small enough to crawl through and open the grate. The rats subsequently owed Jonathan their lives and he continued to work with them while they built their colony. Some months prior to Mrs Brisby's visit to the rose bush, Jonathan had been killed by Fitzgibbons' ornery and fearsome cat, Dragon, whom the rats had been trying to drug. Nicodemus gives Brisby a large red amulet and tells her that it has great supernatural powers when someone shows true courage of the heart. Nicodemus says that the rats will be happy to repay their life debt to Jonathan's family and will move her home.
For the operation to succeed, Dragon will have to be drugged. Mrs Brisby, feeling she should participate, volunteers to spike Dragon's feeding dish. The only way into Fitzgibbons' kitchen is through a small hole under a sideboard, a hole too small for any rat. The plan goes well until Brisby runs back and is trapped by Fitzgibbons' son. Justin leaves her, having to supervise the operation, promising to come back for her. While she's trapped in a bird cage, Brisby overhears Fitzgibbons on the phone with NIMH, who want to come to his farm and capture the rats. Brisby realizes the situation is now more desperate and escapes the bird cage by knocking out it's small water cup.
At the site of the Brisby home, the rats have constructed an elaborate system of ropes and blocks and tackles to lift the cinderblock. While the block is being swung towards the stone, Jenner cuts the anchoring lines with his sword and the block falls, narrowly missing Nicodemus, however, the heavy gears and rope fall on him, killing him. Jenner declares the operation a failure and suggests they leave when Mrs Brisby arrives. Justin tells her that her family is alive but Nicodemus is dead. She suddenly remembers that NIMH will be at the farm tomorrow and tells the rats they must leave tonight. Jenner refuses to believe her and tells the rats that he will take over leadership of the colony. When Mrs Brisby insists she's telling the truth, Jenner strikes her and notices that she's wearing the amulet. Attempting to seize it from her, Justin intervenes and fights with Jenner until he wounds him. Jenner admits that he'd caused the accident that killed Nicodemus, saying that the plan to move the colony would fail. As he moves in to kill Justin, Sullivan kills Jenner with a thrown dagger and succumbs to his own wound, inflicted by Jenner himself.
Moments later, Mrs Brisby sees that her house has begun to sink into the mud, taking her children with it. A futile attempt to rig a new roping system fails and Brisby herself vainly pulls at the remaining ropes as the house sinks under the surface. Justin rescues her, holding her back. As she looks on in horror, the amulet suddenly arises from the mud, brightly glowing, and places itself around her neck. Using the power that Nicodemus spoke of, she raises the house from the mud and moves it to the lee side of the stone. Brisby's children are safe and she faints from exhaustion.
The next morning, with Timmy on the mend from his pneumonia and the rose bush being inspected by NIMH, Jeremy arrives with mounds of string that he'd been collecting to build a love nest and finds the Brisby home already moved and even camouflaged by the natural vegetation. He asks Brisby for the amulet, however, she'd given it to Justin before they'd left the rose bush. While he laments that he wasn't able to help move the house, a female crow suddenly flies into him. The two become immediately infatuated. While Brisby's daughter wraps her mother's hands which were burned by the amulet, Auntie Shrew arrives for a visit, bringing groans from Martin.